Activity of cofilin can be regulated by a mechanism other than phosphorylation/dephosphorylation in muscle cells in culture
Activity of cofilin can be regulated by a mechanism other than phosphorylation/dephosphorylation in muscle cells in culture
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DOI：
10.1007/s10974-007-9117-6
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发表时间：
2007-02-01
影响因子：
2.7
通讯作者：
Obinata, Takashi
中科院分区：
文献类型：
--
作者：
Hosoda, Atsuko;Sato, Naruki;Obinata, Takashi
Cofilin在多种真核细胞中的肌动蛋白丝动力学中起关键作用。其活性通过N-末端侧的Ser 3残基的磷酸化/去磷酸化和/或其与磷酸肌醇PIP 2的结合来调节。为了阐明cofilin活性在肌肉细胞中是如何调节的，我们通过将cofilin的磷酸化位点（Ser 3）分别转化为Ala和Asp来产生非磷酸化形式（A3-cofilin）和磷酸化形式（D3-cofilin）的类似物。这些突变的蛋白质以及具有Ser 3残基的cofilin（S3-cofilin）在E.大肠杆菌表达系统，并与荧光染料缀合。在体外功能测定中，A3-cofilin保留了与F-肌动蛋白结合的能力。在注射到培养的肌细胞中后，A3-cofilin和S3-cofilin迅速破坏细胞质中的肌动蛋白丝，并且产生许多含有外源性cofilin和肌动蛋白的细胞质杆，而D3-cofilin仅在细胞质中扩散而不影响肌动蛋白丝。然而，注射后数小时，A3-cofilin和S3-cofilin的活性受到抑制：肌动蛋白-A3-cofilin（或S3-cofilin）杆消失，cofilin像D3-cofilin一样扩散到细胞质中，肌动蛋白丝重新形成。通过cDNA转染产生的GFP融合的A3-cofilin和S3-cofilin在培养的肌细胞的细胞质中也被抑制。因此，除磷酸化以外的一些机制可以抑制A3-丝切蛋白活性。我们观察到PIP 2可以像结合S3-cofilin一样结合A3-cofilin，并抑制A3-cofilin与肌动蛋白的相互作用。我们的研究结果表明，A3-cofilin和S3-cofilin的活性可以通过PIP 2在肌细胞的细胞质中进行调节。
Cofilin plays a critical role in actin filament dynamics in a variety of eukaryotic cells. Its activity is regulated by phosphorylation/dephosphorylation of a Ser3 residue on the N-terminal side and/or its binding to a phosphoinositide, PIP2. To clarify how cofilin activity is regulated in muscle cells, we generated analogues of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser3) of cofilin to Ala and Asp, respectively. These mutated proteins, as well as the cofilin having Ser3 residue (S3-cofilin), were produced in an E. coli expression system and conjugated with fluorescent dyes. In an in vitro functional assay, A3-cofilin retained the ability to bind to F-actin. Upon injection into cultured muscle cells, A3-cofilin and S3-cofilin promptly disrupted actin filaments in the cytoplasm, and many cytoplasmic rods containing both the exogenous cofilin and actin were generated, while D3-cofilin was simply diffused in the cytoplasm without affecting actin filaments. Several hours after the injection, however, the activity of A3-cofilin and S3-cofilin was suppressed: the actin-A3-cofilin (or S3-cofilin) rods disappeared, the cofilin diffused in the cytoplasm like D3-cofilin, and actin filaments reformed. Both GFP-fused A3-cofilin and S3-cofilin that were produced by cDNA transfection were also suppressed in the cytoplasm of muscle cells in culture. Thus, some mechanism(s) other than phosphorylation can suppress A3-cofilin activity. We observed that PIP2 can bind to A3-cofilin just as to S3-cofilin and inhibits the interaction of A3-cofilin with actin. Our results suggest that the activity of A3-cofilin and also S3-cofilin can be regulated by PIP2 in the cytoplasm of muscle cells.